The present invention relates to methods of using dying cells for treating diseases characterized by pathological immune responses, and to devices for preparing such dying cells. More particularly, the present invention relates to methods of using apoptotic leukocytes for treating diseases characterized by pathological immune responses, such as autoimmune diseases and transplantation-related diseases, and to devices for preparing such apoptotic leukocytes.
Diseases characterized by pathological immune responses include a large number of diseases which are associated with significant mortality and morbidity, and for which no satisfactory/optimal treatments are available. Such diseases particularly include autoimmune diseases, such as systemic lupus erythematosus (SLE), transplantation-related diseases such as graft-versus-host disease (GVHD).
The immune system is a complex network comprising cells, antibodies, tissues, and chemical messenger molecules which allow for communication between these structures. A hallmark of a healthy immune system is the ability to recognize bacteria, viruses, and other foreign bodies and to effectively attack such pathogens while continuing to distinguish between the foreign bodies and the molecules, cells, tissues and organs of the body. In addition to fighting infections, the immune system has other roles in maintaining the normal state of health and function of the body. Throughout the life span of an organism, tissues become reshaped with areas of cells being removed. This is accomplished by a process termed programmed cell death or apoptosis, the apoptotic cells disintegrating in an orderly and harmless fashion and being phagocytosed. In many organs, for example, a certain percentage of the cells die off every day while different branches of the immune system are typically called in to remove the dead cells and parts thereof to make room for the new cells which arise to replace them. Were it not for the cellular debris-removing cells of the immune system, typically macrophages, tissue and organ growth would be impossible due to a lack for space. The process of apoptosis is furthermore considered to be particularly important in the development and maintenance of the immune system itself, where the immune cells which recognize or attack normal cells of the body are destroyed and removed by this process.
The number of monocytes, neutrophils, and lymphocytes that are produced, circulating, dying, and extravasating in the body is controlled at various levels, including via apoptosis.
In the case of monocytes, CFU-GM, the earliest identified cell committed to differentiate along the myeloid pathway, develops into monocyte in the bone marrow, mainly in the presence of M-CSF, IL-3, and low levels of GM-CSF. No bone marrow reserve exists for monocytes, which spend 1-3 days in transit through the marrow and are then released to spend from 8 to 72 hours in the blood, with subsequent further possible differentiation, maturation, and proliferation in tissues [1]. Monocytes comprise 1-6 percent of peripheral leukocytes, and it is estimated that 5.7×106 monocytes/kg are produced every day. Monocytes can survive in tissues as macrophages for long periods, but a substantial portion of monocytes are constantly undergoing apoptosis, either in the absence of anti-apoptotic factors or following infection or activation.
Monocytes express Fas and Fas ligand irrespective of their state of activation [2, 3], and were shown to undergo Fas-dependent apoptosis upon culture [3], activation [4], or infection [5]. Monocytes can be rescued from apoptosis upon exposure to growth factors, differentiating factors (GM-CSF and IL-4), or activation factors [3, 6-8]. Upon differentiation to macrophages, monocytes are rescued from Fas-dependent apoptosis by the expression of Fas-associated death domain-like IL-1-beta-converting enzyme-inhibitory protein (FLIP) [3, 9].
Neutrophils constitute the most abundant population of leukocytes. In humans, the daily turnover of neutrophils is about 1.6×109 cells/kg body weight (Klebanoff S J, Clark R X: The Neutrophil: Function and Clinical Disorders. Amsterdam, North-Holland Publishing, 1978, p 313), which keeps the number of mature neutrophils within defined limits despite the tremendous proliferative potential of the bone marrow precursor cells. This large turnover is mediated by the continuous egress of neutrophils from the circulation. Neutrophils do not return to the circulation but are eliminated by secretion in mucosa or die in the tissues within 1-2 days (Klebanoff S J, Clark R X: The Neutrophil: Function and Clinical Disorders. Amsterdam, North-Holland Publishing, 1978, p 313). Under normal non-inflammatory conditions neutrophil turnover takes place without harmful effects, despite the large bioagressive and destructive potential of these cells displayed under various inflammatory conditions [Weiss S J: Tissue destruction by neutrophils. N Engl J Med 1989; 320:365-376]. A special mechanism of harmless neutrophil destruction is provided by apoptosis, genetically programmed cell suicide.
While apoptosis is a process used by the immune system in protecting the body, it is also used to maintain tolerance to self-antigens and therefore allowing the immune system to fulfill its role in distinguishing the body's own cells from foreign bodies.
Cellular apoptosis plays an important role in antigen-presentation. Immature dendritic cells have the capacity to engulf apoptotic cells and to acquire and immunologically present their antigens Immature dendritic cells that capture apoptotic macrophages exposed to killed influenza-virus, mature and activate lymphocytes to mount virus-specific CTL responses in the presence of conditioned media. However, in the absence of infection and conditioned media, immature dendritic cells do not mature following uptake of apoptotic cells and as a consequence are less able to efficiently present acquired antigens. Furthermore, it has been suggested that following interaction with apoptotic material, immature dendritic cells may have a role in maintaining peripheral tolerance to self-antigens that are permanently created at different sites. In support of this, autoimmunity or SLE-like disease has been observed in mice and humans deficient in receptors important for uptake of apoptotic cells such as ABC1 cassette transporter, Mer, and complement deficiencies, as further described hereinbelow. Clearance via specific receptors may dictate specific immune response or tolerance as demonstrated by TGF-beta and IL-10 secretion by macrophages following uptake of apoptotic cells by macrophages. Thus, cytokines, chemokines, eicosanoids, and additional mediators present in the milieu of the interaction, may polarize the immune response.
When the immune system is deficient in recognition between self- and non-self-antigens, the result is a state of disease, this may result in the immune system attacking one or more specific self molecules or cells leading to tissue and organ damage, resulting in autoimmune disease Immunopathology of non-targeted tissues also may be indirectly caused non-specifically as a consequence of inflammation resulting from immune rejection of neighboring cells and tissues. Other than classical autoimmune diseases such as those mentioned hereinabove, it is becoming increasingly apparent that many vascular disorders, including atherosclerotic forms of such disorders, have an autoimmune component, and a number of patients with vascular disease have circulating autoantibodies. Autoimmune diseases may be divided into two general types, namely systemic autoimmune diseases, such as SLE and scleroderma, and organ specific autoimmune diseases, such as multiple sclerosis, and diabetes. Many clinically different types and subtypes of autoimmune disease occur. Although each type of autoimmune disease is associated with a spectrum of clinical symptoms and aberrant laboratory parameters, signs and symptoms of autoimmune diseases frequently overlap so that one or more are diagnosed in the same patient. The vast majority cases in which one or more autoimmune disease has been diagnosed are characterized by the presence in the affected subject of antibodies directed against self-antigens, termed autoantibodies. Such autoantibodies are often present in tissues at ten to one hundred times the normal level in healthy individuals and give rise to a significant proportion of the organ and tissue damage associated with the particular autoimmune disease. For example, in the autoimmune disease myasthenia gravis, autoantibodies against a receptor in neuromuscular junction are associated with muscle weakness, while in SLE, anti-dsDNA antibodies are associated with nephritis in human patients and can cause nephritis upon injection to normal mice. In such diseases, the tissue and organ damage is attributed to the presence of autoantibodies and to the inflammation, which arises due inflammatory immune responses set off by autoantibodies.
Systemic lupus erythematosus is a model disease for understanding and developing inventive treatments for autoimmune disease in general. While it has long been appreciated that DNA and histones are major autoantigens SLE, only recently has evidence been provided that the DNA-histone complex, i.e., nucleosomes, are the preferred targets of autoantibodies in SLE. During apoptosis, the membrane of cells undergoing apoptosis form cytoplasmic blebs, some of which are shed as apoptotic bodies. It was recently demonstrated that exposure of keratinocytes to high frequency light induces apoptosis, and that the cell surface expression of the ribonucleoproteins Ro and La, but also of nucleosomes and ribosomes, can be explained by translocation of certain intracellular particles to the apoptotic surface blebs. Significantly, another translocation which occurs during apoptosis is that of phosphatidylserine (PS), an acidic phospholipid that normally resides on the inside of the cell, but flips to the outside of the cell membrane when the cell undergoes apoptosis. Phosphatidylserine, like cardiolipin, is a major autoantigen for anti-phospholipid antibodies in SLE. Taken together, these findings suggest that SLE involves autoimmunity directed against intracellular proteins translocated to the cell surface during apoptosis, and hence that SLE patients form an immune response to apoptotic material. This hypothesis is supported by the observation that brief, limited administration of syngeneic apoptotic cells to normal strains of mice leads to induction of autoantibodies and glomerular depositions. The immunopathology of SLE appears to further involve defective uptake of apoptotic material by macrophages, as observed in the reduced uptake/clearance of apoptotic cells by macrophages from SLE patients in-vitro, and by the high incidence of SLE in patients deficient in the C1q and C4 components of the complement system, which is involved in uptake of targeted antigens.
Lymphocytes, i.e. T-cells and B-cells, are relatively resistant to apoptosis. Upon antigenic stimulation, B-cells and T-cells proliferate and some will differentiate into effector cells. Plasma cells secrete antibodies that immobilize pathogens and promote their complement-mediated destruction and Fc (Ig constant region)-receptor-mediated ingestion by certain myeloid cells. Activated T-cells produce cytokines, some of which promote proliferation and functional activation of B-cells and T-cells themselves, whereas others provide feedback signals to cells of the innate immune system Immune effector mechanisms are highly potent weapons designed for the killing of free pathogens and also pathogen-infected host cells. This armory has the potential to destroy healthy cells and tissues because many of the effector molecules, such as pro-inflammatory cytokines, act in a non-antigen-specific manner and also because certain pathogen-specific receptors, such as B-cell receptors (BCRs) and T-cell receptors (TCRs) may cross-react with host antigens.
Immune responses to pathogens therefore pose a potential danger to the host and immunopathology occurs with many types of infection. In addition, chronically activated lymphocytes that are rapidly proliferating, particularly B-cells in germinal centers undergoing Ig-variable gene hyper-mutation, are at risk of sustaining mutations in proto-oncogenes or tumor suppressor genes that could lead to the development of lymphoma and/or leukaemia. Multiple regulatory mechanisms have evolved to prevent immunopathology. These include functional inactivation of cells of the immune system, a process that is potentially reversible and therefore does not eliminate the danger, and killing of no-longer needed and/or potentially dangerous cells by apoptosis [Marsden and A. Strasser, 2003. Annu. Rev. Immunol. 21:71-105].
Cells undergoing apoptosis signal neighboring cells, professional phagocytes, and/or antigen presenting cells to rapidly engulf them, without triggering an inflammatory or autoimmune response [10-12]. This process seems to play an important role in homeostasis, resolution of inflammation, and tolerance induction [13-15]. However disregulation of this process may represent a mechanism of escape from immune surveillance against infections and tumors and, if inefficient, it may support persistent inflammation and autoimmunity [16, 17].
Another issue that remains unclear is the role of apoptotic cell-derived antigens in cross-priming of immune responses. It has been shown that human dendritic cells, but not macrophages, efficiently present antigen that is derived from influenza-infected apoptotic monocytes, which stimulates class I-restricted CD8+ CTLs [18]. It remains unclear how dendritic cells derive a pro-inflammatory presentation of antigens from influenza, since these antigens are acquired from apoptotic cells that are usually considered anti-inflammatory, and that were shown to prevent maturation of dendritic cells [15, 19]. While in the former study conditioned media was employed as an adjuvant, the physiological adjuvants enabling cross-priming nevertheless remain unknown. Thus, antigens derived from apoptotic cells of given lineage may result in of activation or suppression of immunity due to mechanisms which remain to be resolved.
Manipulation of the immune system to treat immunopathology associated with autoimmune diseases, such as SLE, and transplantation-related diseases, such as GVHD, have been major goals of immunologists for many years. Traditionally, such manipulation has involved use of immunosuppressive drugs, such as corticosteroids, azathioprine, cyclophosphamide, and cyclosporine. While such drug-induced immunosuppression has resulted, for example, in improvement of the 5-year survival rate of SLE patients in the last three decades, it is far from being an ideal treatment since no cure is achieved, since such treatment is associated with very serious side-effects, including general immune suppression, leading to high rates of morbidity, and is the primary cause of premature mortality. Administration of biological agents such as anti-CD40 ligand, and CTLA-4Ig has also been advocated. However, the toxicity and efficacy of such treatments is suboptimal, being potentially associated, for example, with general immune suppression similarly to the above-mentioned immunosuppressive drugs.
Thus, in view of the tolerizing/non-inflammatory properties of dying leukocytes described hereinabove, a potentially optimal strategy for treatment of diseases characterized by pathological immune responses, such as autoimmune diseases and transplantation-related diseases, involves administration of dying leukocytes having immunosuppressive/non-inflammatory properties. Such a strategy would inherently circumvent the aforementioned significant disadvantages of prior art immunosuppressive drug-based treatment approaches.
Several prior art approaches involving administration of dying leukocytes have been employed or suggested for treatment of diseases characterized by pathological immune responses.
One approach suggests administration of apoptotic donor cells, such as apoptotic donor leukocytes, to facilitate engraftment of donor hematopoietic grafts transplanted into an allogeneic recipient [Penuche S. et al., 2004. Am J Transplant. 4:1361-5; Kleinclauss F. et al., 2003. Transplantation 75(9 Suppl):43S-45S]. Such an approach, however, suffers from various drawbacks, including requirement for administration of allogeneic leukocytes, which inherently are associated with risk of GVHD as well as of their own rejection, suboptimal efficacy, failure to demonstrate adequate safety with respect to potential for inflammatory side-effects, and/or of never having been attempted in human patients, and hence of never having demonstrated any therapeutic efficacy in human patients.
Another, apheresis-based, approach, termed “extracorporeal photopheresis”, involves administering to a patient a photoactivatable pigment which can be specifically taken up by specific hematopoietic cells, such as T-cells, and following such uptake harvesting blood, isolating the specific hematopoietic cells, triggering their apoptosis via UV irradiation, and infusing them back into the patient (U.S. Pat. No. 6,219,584). This approach has been advocated for treatment of hypersensitivity, graft rejection, or SLE (U.S. Pat. No. 4,838,852); or for amelioration of GVHD. Prior art approaches involving apheresis, however, are often suboptimally effective, and may be associated with undesired side-effects of unknown origin, such as inflammatory side-effects (refer, for example, to: Siami G A. et al., 1997. Cryofiltration apheresis and plasma fractionation causing anaphylactoid reactions in patients receiving angiotensin converting enzyme inhibitors. Ther Apher. 1:325-9; Schwarzbeck A. et al., 1997. Anaphylactoid reactions during dextran apheresis may occur even in the absence of ACE-inhibitor administration. Nephrol Dial Transplant. 12:1083-4; Koga N. et al., 1993. Anaphylactoid reactions and bradykinin generation in patients treated with LDL-apheresis and an ACE inhibitor. ASAIO J. 39:M288-91; Strauss R G., 1996. Mechanisms of adverse effects during hemapheresis. J Clin Apheresis 11:160-4; Rossi P L. et al., 1991. Comparison of the side effects of therapeutic cytapheresis and those of other types of hemapheresis. Haematologica. 76 Suppl 1:75-80; Huestis D W., 1989. Risks and safety practices in hemapheresis procedures. Arch Pathol Lab Med. 113:273-8; Hocker P, Wagner A., 1987. Side-effects of cytapheresis with cell separators. Infusionsther Klin Ernahr 14 Suppl 4:31-5). Extracorporeal photopheresis, in particular, involves generation and administration of harmful necrotic/pro-inflammatory cells (Caricchio R. et al., 2003. Ultraviolet B Radiation-Induced Cell Death: Critical Role of Ultraviolet Dose in Inflammation and Lupus Autoantigen Redistribution. The Journal of Immunology 171:5778-5786).
Thus, all prior art approaches have failed to provide an adequate solution for using dying leukocytes for treatment of diseases characterized by pathological immune responses.
There is thus a widely recognized need for, and it would be highly advantageous to have, a disease treatment method devoid of the above limitation.